The present invention relates to a sample plate, an automated apparatus, a reagent bead or microsphere dispenser, a method of dispensing reagent beads or microspheres, a kit for performing Enzyme Linked Immunosorbent Assay procedures, a kit for performing nucleic acid probe procedures, a method of manufacturing a sample plate and a computer program executable by the control system of an automated apparatus.
An automated reagent bead or microsphere dispenser for dispensing reagent beads or microspheres into a sample plate is disclosed. The sample plate may be used to carry out diagnostic testing such as Enzyme Linked Immunosorbent Assay (“ELISA”) procedures or other immunoassay procedures. Alternatively, the sample plate may be used to carry out testing for DNA or RNA sequences.
In one embodiment, immunoassay procedures are used to test biological products. These procedures can exploit the ability of antibodies produced by the body to recognize and combine with specific antigens which may, for example, be associated with foreign bodies such as bacteria or viruses, or with other body products such as hormones. Once a specific antigen-antibody combination has occurred it can be detected using chromogenic, fluorescent or chemiluminescent materials or less preferably by using radioactive substances. Radioactive substances are less preferred due to environmental and safety concerns regarding their handling, storage and disposal. The same principles can be used to detect or determine any materials which can form specific binding pairs, for example using lectins, rheumatoid factor, protein A or nucleic acids as one of the binding partners.
In one embodiment, ELISA is a form of immunoassay procedure used, wherein one member of the binding pair is linked to an insoluble carrier surface (“the solid phase”) such as a sample vessel, and after reaction the bound pair is detected by use of a further specific binding agent conjugated to an enzyme (“the conjugate”). The characteristics and choice of solid phases for capture assays, on methods and reagents for coating solid phases with capture components, on the nature and choice of labels, and on methods for labeling components is well known in the arts and may also be applied to assays for other specific binding pairs. An example of a standard textbook is “ELISA and Other Solid Phase Immunoassays, Theoretical and Practical Aspects”, Editors D. M. Kemeny & S. J. Challacombe, published by John Wiley, 1988. Such advice.
In one embodiment of ELISA, the solid phase is coated with a member of the binding pair. An aliquot of the specimen to be examined can incubated with the solid coated solid phase and any analyte that may be present is captured onto the solid phase. After washing to remove residual specimen and any interfering materials it may contain, a second binding agent, specific for the analyte and conjugated to an enzyme can be added to the solid phase. During a second incubation any analyte captured onto the solid phase can combine with the conjugate. After a second washing to remove any unbound conjugate, a chromogenic substrate for the enzyme can be added to the solid phase. Any enzyme present can begin converting the substrate to a chromophoric product. After a specified time the amount of product formed may be measured using a spectrophotometer, either directly or after stopping the reaction.
Many variants are known in the art including fluorogenic and luminogenic substrates for ELISA, direct labeling of the second member of the binding pair with a fluorescent or luminescent molecule (in which case the procedure is not called an ELISA but the process steps are very similar) and nucleic acids or other specific pairing agents instead of antibodies as the binding agent. In some embodiments, the assays use fluid samples, e.g. blood, serum, urine, etc., which are aspirated from a sample tube and are then dispensed into a solid phase. Samples may be diluted prior to being dispensed into the solid phase or they may be dispensed into deep well microplates, diluted in situ and then the diluted analyte may be transferred to the functional solid phase.
In one embodiment, the solid phase is a standard sample vessel known as a microplate, which can be stored easily and which may be used with a variety of biological specimens. The microplate can be made from materials including, but not limited to polystyrene, PVC, Perspex or Lucite. In one embodiment, the microplate measures approximately 5 inches (12.7 cm) in length, 3.3 inches (8.5 cm) in width, and 0.55 inches (1.4 cm) in depth. In one embodiment, the microplate is made from polystyrene wherein the polystyrene's enhanced optical clarity assists visual interpretation of the results of a reaction. The polystyrene microplate can also be compact, lightweight and easily washable. In one embodiment, the microplate is sold under the name “MICROTITRE”®. The microplate can comprise 96 wells (also known as “microwells”) which can be symmetrically arranged in an 8×12 array. The microwells can have a maximum volume capacity of approximately 350 μl. In one embodiment, approximately 10-200 μl of fluid is dispensed into a microwell. In some arrangements of the microplate the microwells may be arranged in strips of 8 or 12 wells that can be moved and combined in a carrier to give a complete plate having conventional dimensions.
Positive and negative controls can be supplied with microplates, such as with commercial kits, and are used for quality control and to provide a relative cut-off. After reading the processed microplate, the results of the controls can be checked against the manufacturer's validated values to ensure that the analysis has operated correctly and then the value is used to distinguish positive from negative specimens and a cut off value is calculated. Standards can be provided for quantitative assays and used to build a standard curve from which the concentration of analyte in a specimen may be interpolated.
In one embodiment, the ELISA procedure can involve multiple steps including, but not limited to, one or more of the following: pipetting, incubation, washing, transferring microplates between activities, reading and data analysis. One or more of the steps (or “phases”) involved in the ELISA procedures such as sample distribution, dilution, incubation at specific temperatures, washing, enzyme conjugate addition, reagent addition, reaction stopping and the analysis of results, can be automated. For example, the pipette mechanism used to aspirate and dispense fluid samples uses disposable tips which are ejected after being used so as to prevent cross-contamination of patients' samples. Multiple instrumental controls can be in place to ensure that appropriate volumes, times, wavelengths and temperatures are employed, data transfer and analysis is fully validated and monitored. Automated immunoassay apparatus for carrying out ELISA procedures can be used in laboratories of e.g. pharmaceutical companies, veterinary and botanical laboratories, hospitals and universities for in-vitro diagnostic applications such as testing for diseases and infection, and for assisting in the production of new vaccines and drugs.
ELISA kits can comprise microplates having microwells which have been coated with a specific antibody (or antigen). For example, in the case of a hepatitis B antigen diagnostic kit, the kit manufacturer will dispense anti-hepatitis B antibodies which have been suspended in a fluid into the microwells of a microplate. The microplate is then incubated for a period of time, during which time the antibodies adhere to the walls of the microwells up to the fluid fill level (typically about half the maximum fluid capacity of the microwell). The microwells are then washed leaving a microplate having microwells whose walls are uniformly covered with anti-hepatitis B antibodies up to the fluid fill level.
A testing laboratory can receive a number of sample tubes containing, for example, body fluid from a number of patients. A specified amount of fluid can be aspirated out of the sample tube using a pipette mechanism and dispensed into one or more microwells of a microplate that has been previously prepared by the manufacturer, such as discussed above. If it is desired to test a patient for a number of different diseases, fluid from the patient is typically dispensed into a number of separate microplates, wherein each microplate may have been coated by its manufacturer with a different binding agent. Each microplate can then be processed separately to detect the presence of a different disease. This can lead to analysis of several different analytes with a multiplicity of microplates and transfer of aliquots of the same specimen to the different microplates, resulting in large numbers of processing steps and incubators and washing stations that can cope with many microplates virtually simultaneously. In automated systems this instruments may have multiple incubators and complex programming to avoid clashes between microplates with different requirements. For manual operation either several technicians may be needed or the throughput of specimens is slow. It is possible to combine strips of differently coated microwells into a single carrier, add aliquots of a single specimen to the different types of well and then perform the ELISA in this combined microplate. Constraints on assay development, however, can make this combination difficult to achieve and can lead to errors of assignment of result, while manufacture of microplates with several different coatings in different microwells can present difficulties of quality control.
Conventional ELISA techniques have typically concentrated upon performing the same single test upon a plurality of patient samples per microplate or in detecting the presence of one or more of a multiplicity of analytes in those patients without distinguishing which of the possible analytes is actually present. For example, it is commonplace to determine in a single microwell whether a patient has antibodies to HIV 1 or HIV 2, or HIV 1 or 2 antigens, without determining which analyte is present and similarly for HCV antibodies and antigens.
However, a new generation of assays are being developed which enable multiplexing to be performed. Multiplexing enables multiple different tests to be performed simultaneously upon the same patient sample.
In one embodiment, multiplexing provides a microplate comprising 96 sample wells wherein an array of different capture antibodies is disposed in each sample well. The array can comprise an array of 20 nL spots each having a diameter of 350 μm. The spots can be arranged with a pitch spacing of 650 μm. Each spot corresponds with a different capture antibody.
Multiplexing enables a greater number of data points and more information per assay to be obtained compared with conventional ELISA techniques wherein each sample plate tests for a single analyte of interest. The ability to be able to combine multiple separate tests into the same assay can lead to considerable time and cost savings. Multiplexing also enables the overall footprint of the automated apparatus to be reduced.
Provided herein is a sample plate and associated automated apparatus which has an improved format and which provides a greater flexibility.
In addition to ELISA procedures, a hybridization probe can be used to test for the presence of DNA or RNA sequences. A hybridization probe typically comprises a fragment of nucleic acid, such as DNA or RNA, which is used to detect the presence of nucleotide sequences which are complementary to the nucleic acid sequence of the probe. The hybridization probe can hybridize to single-stranded nucleic acid (e.g. DNA or RNA) whose base sequence allows pairing due to complementarity between the hybridization probe and the sample being analyzed. The hybridization probe may be tagged or labeled with a molecular marker such as a radioactive or more preferably a fluorescent molecule. The probes are inactive until hybridization at which point there is a conformational change and the molecule complex becomes active and will then fluoresce (which can be detected under UV light) DNA sequences or RNA transcripts which have a moderate to high sequence similarity to the probe are then detected by visualizing the probe under UV light.
It is desired to provide an improved sample plate for retaining reagent beads, as well as related systems and methods.